The baculovirus expression vector system (BEVS) has gained acceptance as the system of choice for the expression of foreign genes of both prokaryotic and eukaryotic origin (V. A. Luckow & M. D. Summers, Biotechnology, Vol. 6, 47-55, 1988). This expression system is one of the easiest and safest eukaryotic systems for recombinant protein production. A number of genes of both prokaryotic and eukaryotic origin have been expressed using this system. BEVS primarily utilizes the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the prototype baculovirus which infects Lepidopteran insects. On the basis of their temporal activation profile in the infected insect host, baculovirus genes are classified as early, late and very late (D. R. O'Reilly, et al., In: Baculovirus Expression Vectors: A Laboratory Manual, New York: W H Freeman and Co, 1992). For the purpose of foreign gene expression, promoters that drive the transcription of late and very late genes are most commonly used. These include the late cor promoter and the very late p10 and polyhedrin gene promoters.
The polyhedrin promoter, utilized extensively in BEVS is temporally regulated and hyperactive (V. S. Luckow & M. D. Summers, Biotechnology, Vol. 6, 47-55, 1988: M. D. Summers, Curr. Commun. in Molec. Biol., Cold Spring Harbor Press. Cold Spring Harbor, N.Y. 1987; M. D. Summers & G. E. Smith, TAES Bulletin No. 1555, 1988; Smith and Summers, U.S. Pat. Nos. 4,745,051 and 4,040,367; M. D. Summers et al. 5,169,784; Webb & Summers, vol. 2, Technique--A Journal of Methods in Cell and Molec. Biology, 173-188, 1990).
There are eight interspersed homologous regions (hrs) that vary in size from 30 to 800 bp distributed in the double-stranded, covalently closed baculovirus DNA genome (M. A. Cochran & P. Faulkner, Vol. 45, J. Virology, 961-970,1983; Kool, et al. Vol. 74, J. General Virology, 2661-2668, 1993; S.M. Rodems & P. D. lriesen, Vol. 69, J. Virology, 5365-5378, 1995). These are designated as hrl, hrla, hr2, hr3, hr4a, hr4b, hr4c and hr5. The sequence homology between these regions is due to the presence of conserved 30 bp palindromic repeats (L. A. Guarino et al. Vol. 60, J. Virology, 224-229, 1986; L. A. Guarino & M. D. Summers. Vol. 60, J. Virology 215-223, 1986; A. Liu et al. Vol. 67, J. General Virology, 2565-2570, 1986). The homologous region sequences are capable of functioning as origins of replication (ori) (M. Pearson, et al. Vol. 257, Science, 1382-1384, 1992; M. Kool et al. Vol. 192, Virology, 94-101, 1993; M. Kool et al. Vol. 198, Virology, 680-689, 1994; C.H. Ahrens, et al. Baculovirus DNA replication, DePamphilis, M. (ed.) In: DNA replication in eukaryotes, 1995; M. Kool, et al. Vol. 76, J. General Virology, 2103-2118, 1995; D. J. Leisy & G. F. Rohrmann, Vol. 196, Virology, 722-730, 1993) as well as enhancers of some viral genes (S. M. Rodems & P. D. Friesen, Vol. 69, J. Virology, 5365-5378, 1995; L. A. Guarino, et al. Vol. 60, J. Virology, 224-229, 1986; L. A. Guarino & M. D. Summers, Vol. 60, J. Virology, 215-223, 1986; D. D. Carson et al. Vol. 65, J. Virology, 945-951, 1991; A. Lu & E. B. Carstens, Vol. 195, Virology, 710-718, 1993; M. S. Nissen, & P. D. Friesen, Vol. 63, J. Virology, 493-503, 1989; S. M. Rodems & P. D. Friesen, Vol. 67, J. Virology, 5776-5785, 1993; L. A. Guarino & W. Dong, vol 65, J. Virology, 3676-3680, 1991; L. A. Guarino, & W. Dong, Vol. 200, Virology, 328-335, 1994). The hr5, in particular, has been demonstrated to function as an enhancer of the delayed early 39K gene (S. M. Rodems & P. D. Friesen. Vol. 69, J. Virology, 5365-5378, 1995). The applicants previously demonstrated that the hr1 homologous region sequence, located about 3.7 kb upstream to the polyhedrin promoter in the wild type AcMNPV genome, enhances transcription from the polyhedrin promoter in a position- and orientation-independent manner in transient expression assays (S. Habib et al. Vol. 15, DNA Cell Biology, 737-747, 1996). Enhancement of expression was demonstrated to be a direct result of enhanced transcription from the promoter and was independent of the ori function of hr1.
The property of viral enhancers has not been exploited in the baculovirus expression vector system. Specifically, hr1 has not been exploited in baculovirus expression plasmids or baculovirus expression vectors for enhanced foreign gene expression in insect cells.
It is an object of this invention to produce expression vector plasmids that can be used to construct viral expression vectors or that can be used to tra isfect cells to enhance the expression of foreign genes in insect cells.
It is an object of this invention to produce a baculovirus expression vector to enhance the expression of foreign genes in insect cells.
It is a further object of this invention to construct a recombinant baculovirus expression plasmid that can be used to construct a baculovirus expression vector which can be used to increase the yield of the desired protein product or protein products.
It is another object of this invention to construct a recombinant baculovirus enhancer of foreign gene expression driven by the baculovirus polyhedrin promoter.
It is yet another object of the invention to construct a recombinant baculovirus enhancer of foreign gene expression driven by the Drosophila hsp 70 promoter.
A still further object of the invention is to construct a recombinant baculovirus expression vector comprising hrl as a transcriptional enhancer of foreign gene expression driven by the baculovirus polyhedrin gene promoter.
Yet another object of this invention is an approach for the construction of a recombinant baculovirus expression vector comprising hrl as a transcriptional enhancer of foreign gene expression driven by the Drosophila hsp 70 promoter.
Still another object of this invention is an approach for the construction of a recombinant baculovirus expression vector comprising hrl as a transcriptional enhancer of the expression of one or more foreign genes driven by the baculovirus polyhedrin gene promoter and/or Drosophila hsp 70 promoter.
Yet another object of this invention is an approach for the construction of a recombinant baculovirus expression vector comprising two hr1 sequences as transcriptional enhancers of the expression of one or more foreign genes under the control of multiple promoters such as two polyhedrin promoters, two Drosophila hsp 70 promoters or a polyhedrin promoter and a Drosphila hsp 70 promoter.